RESUMEN
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63-69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.
Asunto(s)
Antígenos de Neoplasias , Proteínas Ligadas a GPI , Inmunoconjugados , Proteínas de Neoplasias , Neoplasias de la Próstata , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/inmunología , Inmunoconjugados/farmacología , Animales , Antígenos de Neoplasias/inmunología , Ratones , Proteínas Ligadas a GPI/inmunología , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Línea Celular Tumoral , Oligopéptidos/inmunología , Oligopéptidos/farmacología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacologíaRESUMEN
Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer. Recently, the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (known as CEA or CD66e) was suggested to be a specific surface protein marker for NEPC. Therefore, we identified a new, fully-human anti-CEACAM5 monoclonal antibody, 1G9, which bound to the most proximal membrane domains, A3 and B3, of CEACAM5 with high affinity and specificity. It shows no off-target binding to other CEACAM family members, membrane distal domains of CEACAM5, or 5800 human membrane proteins. IgG1 1G9 exhibited CEACAM5-specific ADCC activity toward CEACAM5-positive prostate cancer cells in vitro and in vivo. Chimeric antigen receptor T cells (CAR-T) based on scFv 1G9 induced specific and strong antitumor activity in a mouse model of prostate cancer. Our results suggest that IgG1 and CAR-T cells based on 1G9 are promising candidate therapeutics for CEACAM5-positive NEPC and other cancers.
Asunto(s)
Antígeno Carcinoembrionario/genética , Tumores Neuroendocrinos/terapia , Neoplasias de la Próstata Resistentes a la Castración/terapia , Neoplasias de la Próstata/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/inmunología , Antígeno Carcinoembrionario/inmunología , Antígeno Carcinoembrionario/uso terapéutico , Proliferación Celular/efectos de los fármacos , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina G/inmunología , Inmunoterapia Adoptiva/tendencias , Masculino , Ratones , Tumores Neuroendocrinos/inmunología , Tumores Neuroendocrinos/patología , Próstata/patología , Próstata/cirugía , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/inmunología , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.
Asunto(s)
Prueba Serológica para COVID-19/métodos , Vacunas contra la COVID-19/inmunología , COVID-19/terapia , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Prueba Serológica para COVID-19/normas , Vacunas contra la COVID-19/normas , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Inmunización Pasiva/métodos , Inmunización Pasiva/normas , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero , Sueroterapia para COVID-19RESUMEN
Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized replication competent SARS-CoV-2 with exceptional potency as measured by two different assays. There was no enhancement of pseudovirus infection in cells expressing Fcγ receptors at any concentration. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 potently neutralized mouse ACE2 adapted SARS-CoV-2 in wild type BALB/c mice and native virus in hACE2 expressing transgenic mice. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.
RESUMEN
The IgG1 CH2 domain is involved in Fc-mediated effector functions and is a promising scaffold for development of novel therapeutics. We previously reported that removal of seven unstructured N-terminal residues of an autonomous human IgG1 CH2 domain significantly increased its stability and aggregation resistance. However, the way in which the C-terminal residues affect folding is unclear. Here, we found that the CH2 C-terminus is highly sensitive to truncation although these residues adopt a random coil conformation according to the crystal structure of a CH2 domain. To optimize the C-terminus, we used a phage display platform for high-throughput screening of mutants with improved physicochemical properties. After panning of the CH2 mutant library at high temperature against a CH2-specific antibody recognizing a conformational epitope, we obtained two candidates, B3 and D9, with markedly increased thermal stability. We found that substitution of K338 (EU numbering) by isoleucine is crucial for the increased stability, which might be due to enhanced hydrophobic interactions involving W313. However, the aggregation propensity was also increased. To reduce the aggregation propensity, we further mutated the last two residues A339 and K340 adjacent to residue I338 at the C-terminus by rational design and identified a mutant, CH2-IKS (K338I, A339K, and K340S), with high stability and aggregation resistance. In summary, the C-terminus of CH2 is important for its folding and could be further optimized toward better potential applications for CH2-based therapeutics. Our strategy might be also useful for stabilization of other Ig-like proteins.